ARTICLE
High-Throughput Lensfree Imaging and
Characterization of a Heterogeneous Cell Solution
On a Chip
Ting-Wei Su,1 Sungkyu Seo,1 Anthony Erlinger,1 Aydogan Ozcan1,2,3
1
Electrical Engineering Department, University of California, P.O. Box 951594, Los Angeles,
California 90095; telephone: 310-825-0915; fax: 310-206-4833; e-mail: ozcan@ucla.edu
2
Biomedical Engineering IDP, University of California, Los Angeles, California
3
California NanoSystems Institute (CNSI), University of California, Los Angeles, California
Received 4 May 2008; revision received 27 August 2008; accepted 2 September 2008
Published online 8 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22116
ABSTRACT: A high-throughput on-chip imaging platform
that can rapidly monitor and characterize various cell types
within a heterogeneous solution over a depth-of-field of
4
mm and a field-of-view of
10 cm2 is introduced. This
powerful system can rapidly image/monitor multiple layers
of cells, within a volume of
4 mL all in parallel without the
need for any lenses, microscope-objectives or any mechan-
ical scanning. In this high-throughput lensless imaging
scheme, the classical diffraction pattern (i.e., the shadow)
of each micro-particle within the entire sample volume is
detected in less than a second using an opto-electronic
sensor chip. The acquired shadow image is then digitally
processed using a custom developed ‘‘decision algorithm’’ to
enable both the identification of the particle location in 3D
and the characterization of each micro-particle type within
the sample volume. Through experimental results, we show
that different cell types (e.g., red blood cells, fibroblasts, etc.)
or other micro-particles all exhibit uniquely different sha-
dow patterns and therefore can be rapidly identified without
any ambiguity using the developed decision algorithm,
enabling high-throughput characterization of a heteroge-
neous solution. This lensfree on chip cell imaging platform
shows a significant promise especially for medical diagnostic
applications relevant to global health problems, where com-
pact and cost-effective diagnostic tools are urgently needed
in resource limited settings.
Biotechnol. Bioeng. 2009;102: 856–868.
ß 2008 Wiley Periodicals, Inc.
KEYWORDS: on-chip imaging; lensfree—lensless imaging;
high-throughput cytometry; cell counting; point-of-care
devices; global health
Ting-Wei Su and Sungkyu Seo contributed equally to this study.
Correspondence to: A. Ozcan
856 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
Introduction
Rapid and high-throughput counting and characterization
of different cell types within a heterogeneous mixture is a
challenging, but an important task for various applications
in bioengineering and medicine. In clinical settings, the cell
count measurements are mostly performed by using flow-
cytometry, which is expensive, complex and bulky (Burger
and Gershman, 1988; Darzynkiewicz et al., 1999; Hulett
et al., 1973; Kamentsky et al. 1997). Furthermore, the
characterization speed of most flow-cytometers is practically
less than 10,000 cells/s, which sets a certain limit on their use
towards high-throughput screening. There have been
various studies to miniaturize conventional bench-top
flow-cytometers into portable units. However, rather than
increasing the throughput, these efforts have been mostly
focused on developing techniques that can provide higher
detection efficiency, reduced size, or reduced stress to
vulnerable cells (Kamei et al., 2003; Lancaster et al., 2005;
Niehren et al., 1995; Novak et al., 2007; Wolff et al., 2003).
For instance, a rapid electro-osmotic micro pump in a
vertical channel only allows a controlled flow speed of 120
cells/min (Joo et al., 2007). Furthermore, most of such
micro-cytometers that used charged coupled device (CCD)
or complementary field oxide semiconductor (CMOS)
cameras for optical detection were limited to a flow rate of
less than 100 cells/sec, which is significantly slower than
conventional flow-cytometry (Lien et al., 2005). In the
meantime, for point-of-care applications, there is an urgent
need for a compact, cost-effective and high-throughput cell
counting/imaging device that can possibly work even at
resource limited settings, with minimum amount of training
(Cheng et al., 2005, 2007; Chin et al., 2006; Macnab and
Koshland, 1972; Lee, 2003; Ozcan et al., 2007).
For high-throughput cell counting or monitoring
applications, a high resolution optical microscope (see
e.g., Ozcan, 2006; Steinberg and Ali, 2001; Weinstein et al.,
ß 2008 Wiley Periodicals, Inc.
2004) is not always necessary. The most relevant informa-
tion that a high-throughput cell characterization system
should provide is the type and the count of the cells of
interest within the sample volume. Recently, we proposed an
imaging platform termed ‘‘Lensless, Ultra wide-field Cell
monitoring Array platform based on Shadow imaging’’
(LUCAS), which relies on recording the shadow image of
each individual cell onto an optoelectronic sensor array
plane (Ozcan and Demirci, 2007). In LUCAS, the cells of
interest are positioned on a microscope slide or within a
microfluidic device, and are uniformly illuminated with a
regular incoherent white-light source. After a diffraction
limited propagation of for example, 0.1–0.2 mm through the
bottom substrate, the shadow pattern of each cell is digitally
recorded using a sensor array such as a charge-coupled
device (CCD) as shown in Figure 1a.
This initial demonstration of LUCAS could handle a
limited sample solution volume of <0.08 mL, and was only
applicable to homogenous cell solutions, which can be
considered as important limitations. In this manuscript, we
significantly expand this previous technique in two major
aspects:
(1) We experimentally illustrate that the same LUCAS
platform can significantly be improved to enable unique
identification and characterization of various cell types
or other microscopic particles within a heterogeneous
cell mixture using a custom-developed ‘‘decision
algorithm’’. This novel algorithm processes the unique
diffraction pattern corresponding to each micro-
particle/cell to identify its type and its three-dimen-
sional location within the sample volume.
(2) We also significantly improve the depth-of-field and the
sample volume that can be monitored using LUCAS.
Specifically, we illustrate that, unlike a conventional
optical microscope, the LUCAS platform can monitor
and characterize cells/micro-particles over an ultra-large
depth-of-field of
4 mm without the need for
refocusing. Combined with its ultra-wide field-of-view
of
10 cm2, this implies rapid and unambiguous

Figure 1. a: Experimental LUCAS set-up illustrating the effect of diffraction on the cell shadow; (b) Single frame LUCAS image for 3T3 cells that is captured using the set-up of
(a) over an ultra-wide field of view of
10 cm2. The insets show the zoomed images of various points on the LUCAS image illustrating the diffraction signatures of individual 3T3 cells.
The same figure with white dashed circles also shows the field-of-view of 10 and 5 objective-lenses. This figure demonstrates that LUCAS can monitor a field-of-view that is
2
orders of magnitude wider than a regular optical microscope. c: A brief illustration of LUCAS imaging steps. [Color figure can be seen in the online version of this article, available at
www.interscience.wiley.com.]

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Biotechnology and Bioengineering
 characterization and localization of micro-particles, pipette between two microscope slides or within a liquid
including cells, within a heterogeneous sample volume reservoir (e.g., a micro-fluidic channel) which has an area of
of
4 mL. 10 cm2. Then, the entire sample (together with the
substrate) is positioned onto the active region of the digital
Therefore, we can conclude that not only the biological sensor array using a vacuum pen (NT57-636, Edmund
relevance of our previous technique (Ozcan and Demirci, Optics, Barrington, NJ). Illuminated by a collimated beam
2007) has been significantly improved by being able to work of an incoherent light source for example, a monochromator
with a heterogeneous cell mixture through the use of a novel (Oriel CornerstoneTM 260-1/4 m, Newport) or a fiber optic
decision algorithm, but also the sample volume that is white-light illuminator (DC-950, Dolan-Jenner, Boxborough,
imaged has been increased by more than 40-fold. This MA), the shadow/diffraction signature of each micro-object
powerful cell imaging platform does not use any lenses or falls onto the sensor array forming the LUCAS image.
microscope objectives, and therefore offers an extremely
compact device volume that can eventually be integrated
within a regular cell-phone. Given its compact design and
Decision Algorithm
ultra large sample volume, combined with a powerful digital
processing interface, this on-chip imaging platform holds For the development of LUCAS cell identification and
great promise for point-of-care diagnostic devices that can characterization algorithm, some of the useful quantitative
be used to rapidly measure the count of for example, red image metrics, among many others, are the diameter, the 2D
blood cells (RBCs) or T-lymphocytes (such as CD4 cells) texture, and the contrast of the LUCAS shadow image with
from whole blood samples, especially in resource limited respect to the background. Figure 2 illustrates the unique
settings. diffraction signatures of several cell lines or other micro-
particles imaged using LUCAS. The developed decision
algorithm functions based on primarily three parameters: (i)
Materials and Methods A threshold level, which defines the intensity level of the
micro-object signature compared to the background noise
Sample Preparation and High-Throughput level; (ii) The width of the target micro-object in terms
On Chip Imaging of number of pixels; and (iii) the 2D correlation of the
To illustrate the unique capability of the LUCAS system, statistical signature of a target object type with the acquired
Figure 1b shows a single frame LUCAS image for NIH-3T3
cells (fibroblasts; suspended within 1 phosphate buffered
saline (PBS) solution, that is, non-adherent to the substrate)
that is acquired within less than 1 s using the set-up of
Figure 1a over a field of view of
10 cm2. This raw LUCAS
image has a digital size of >20 MB and is composed of >11
million pixels. For this experiment we used a CCD chip with
a pixel size of 9 mm  9mm having an active area of
10 cm2
(KAI-11002, Kodak, Rochester, NY). The cover glass of the
CCD chip was removed to better control the object-to-
sensor distance, for example, 100 mm in Figure 1b. The
electronic shutter was adjusted to capture one LUCAS frame
with
438 ms integration time, which roughly constitutes
half of the image read-out time. Note that since there is no
external fluid flow during this integration time window,
the diffraction signature of the cells within the solution is
not affected. The insets of Figure 1b also show the zoomed
images of various points on the LUCAS image illustrating
the unique diffraction signatures of individual 3T3 cells.
The same figure also shows with white dashed circles the
field-of-view of standard 10 and 5 objective-lenses. This
figure demonstrates that the LUCAS platform can monitor a
field-of-view that is
2 orders of magnitude wider than a
regular optical microscope, highlighting its high-throughput
nature. Figure 2. For LUCAS images, the shape and the contrast difference among
Figure 1c briefly illustrates the on chip imaging process in different cell types and micro-beads is shown. In these lensfree LUCAS images, the
LUCAS. Initially, the micro-objects (such as cultured cells of contrast is reversed for cells versus micro-particles. Furthermore, each cell type
exhibits a characteristic LUCAS signature that permits digital sorting of different cell
interest) are suspended in for example, 1 PBS solution, types from each other within a LUCAS image.
and this diluted sample solution is placed using a micro-

858 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
LUCAS image of interest. These parameters were robust
enough to accurately characterize heterogeneous mixtures
of various micro-objects such as RBCs, hepatocytes, yeast
cells and micro-beads. The user-interface together with a
simplified flow-chart of this developed algorithm are
illustrated in Figure 3a and b, respectively. We illustrate
in the screen-shot image of Figure 3a, computer-based
analysis of a LUCAS image for a two-layered heterogeneous
mixture of 10 mm beads, RBCs, and yeast cells (S. Pombe).
Each layer in this solution is separated by
2 mm. In this
figure, the location and the height of each RBC, yeast cell,
and micro-bead is automatically detected using a custom-
developed decision algorithm, which matches the unique
statistical diffraction signature of any micro-object with the
thresholded LUCAS image for identification and localiza-
tion of each target cell/particle.
For 2D correlation calculations, the decision algorithm
utilizes a set of training LUCAS images corresponding to
homogenous populations in order to build a statistical
image library for each cell/micro-object type at a given
depth-of-field. Using this statistical approach, the effect of
the variations in the shadow images of the same cell kind on
the accuracy of the counting algorithm can be minimized.
To provide an example of how such 2D correlation maps are
utilized in LUCAS, in Figure 4 we illustrate the details of
LUCAS characterization for a heterogeneous mixture of
RBCs, yeast cells (S. Pombe) and 3 mm diameter polystyrene
beads imaged at a DOF of S ¼ 625 mm. In Figure 4b, we
show a zoomed region of the acquired LUCAS image and its
comparison with a regular 10 microscope image (Fig. 4c).
Figure 4d shows the output of our decision algorithm,
marking with different colored circles each cell/particle type.
For comparison purposes the 10 microscope image in
Figure 4c also uses the same colored circles for each micro-
object. In our characterization results, except the diffraction
signature that is pointed with a triangle in Figure 4d, all the
characterization decisions are correct, and exactly match
with the microscope image. For the triangular spot in
Figure 4d, there is actually a cluster of 2 microbeads together
with a yeast cell, all touching each other, which forced an
error in our decision algorithm as marked in Figure 4d—see
the microscope image in Figure 4c. This error is unavoidable
in LUCAS since the diffraction pattern of the clustered
object is uniquely different than the individual micro-
objects making up the cluster. To further provide the details
of these characterization results, we show in Figure 5 the
2D correlation maps of each micro-object type that are
calculated using the LUCAS library image of the target cell/
particle imaged at the same DOF value of S ¼ 625 mm and
under the same illumination conditions. Figure 5 clearly
indicates that for RBCs, yeast cells and the 3 mm beads, each
2D correlation map exhibits a strong peak at the locations
where the target diffraction signatures reside (compare
Fig. 4c and d with Fig. 5). Furthermore, it is worth noting
that the LUCAS decision algorithm was quite robust for
some of the partially overlapping diffraction signatures: see
for example, Fig. 5b and c for individual yeast or 3 mm bead
diffraction signatures that were partially overlapping with
other diffraction signatures and could still be correctly
identified in their corresponding 2D correlation maps.
Due to its ultra-wide FOV of
10 cm2, this decision
algorithm can in principle identify and characterize
50,000
cells within 1 s over this entire FOV of the LUCAS platform.
In general, for a single plane of cells, if there exists M cells
within the entire LUCAS field-of-view, and each cell has a
shadow diameter of CS, then the percentage of cells that do
not overlap can be written as exp(pM(CS)2/FOV), where
FOV (the field-of-view of the LUCAS platform) has been
assumed to be large when compared to M(CS)2. This implies
that for M ¼ 50,000; CS ¼ 25 mm; and a FOV of
10 cm2,
>90% of the imaged cells will not be overlapping. As a
matter of fact, some portion of the remaining
10% of the
‘‘statistically overlapping’’ cells can still be counted by using
powerful segmentation and edge detection algorithms,
reducing the statistical error further below 10%. Therefore,
the statistical error rate of the automated decision algorithm
due to cell overlap probability will not be a limiting factor
for less than 50,000 cells over a LUCAS FOV of
10 cm2.
Our initials studies indicate that the accuracy of our
decision algorithm varies based on the mixture of micro-
objects to be analyzed. With homogenous mixtures
containing only beads, RBCs, fibroblasts, etc. a count
accuracy as high as >98% was easily achieved. We also
achieved a count accuracy of over
90% for artificial
mixtures of for example, RBCs, hepatocytes or fibroblasts
imaged at a DOF of <500 mm. Meanwhile, for hetero-
geneous solutions imaged at a longer DOF, for example, >2
mm, the characterization results based on the raw LUCAS
image can sometimes yield an unacceptable error rate of for
example, >30%. To better combat such characterization
issues at longer DOF values of >2 mm, use of digital noise
reduction filters (e.g., Enhanced Lee Filter (Lee, 1981)) and
more complex illuminations schemes using for example,
multiple-wavelengths are currently being explored.
Results
Since LUCAS is a lensless system, there is no optical
magnification and the spatial resolution of the recorded
diffraction signatures is quite poor (i.e., under-sampled).
This, however, is not a limitation for LUCAS since the
diffraction signatures corresponding to different micro-
scopic objects exhibit uniquely different 2D textures. This
characteristic shadow signature of each micro-object type
enables automated counting and characterization of various
particles/cells within a heterogeneous solution even with
highly pixelated low resolution LUCAS images. Our initial
results illustrating this unique cell identification/character-
ization potential of the LUCAS platform are shown in
Figure 2 for red blood cells (RBCs), fibroblasts, hepatocytes,
mouse embryonic stem cells (mES) and polystyrene micro-
beads. First, note that in these lensfree LUCAS diffraction
images, the contrast is reversed for micro-beads versus cells,
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Figure 3. a: LUCAS characterization results of a two-layered heterogeneous mixture are illustrated. Each layer of the mixture, located at S ¼ 1.0 mm and S ¼ 3.0 mm, contains
10 mm diameter micro-beads, red blood cells, and yeast cells (S. Pombe), all suspended in 1 PBS solution. Different colored solid and dashed circles mark the location and the
height of each cell/particle type located within the sample volume—see the figure legend. b: Simplified flow-chart of the decision algorithm is shown. [Color figure can be seen in
the online version of this article, available at www.interscience.wiley.com.]

860 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
Figure 4. a: A LUCAS characterization experiment is illustrated for a heterogeneous mixture of RBCs, yeast cells and 3 mm beads imaged at a DOF of 625 mm. b: LUCAS image
showing a zoomed region of interest. c: 10 microscope image of the same FOV that is imaged in (b). d: Automated LUCAS characterization results marking the location of each cell/
object type. For comparison purposes the 10 microscope image in (c) also uses the same colored circles for each micro-object type. In our characterization results, except the
diffraction signature that is pointed with a triangle in (d), all the characterization decisions are correct, exactly matching with the microscope image. For the triangular spot, there is
actually a cluster of two microbeads together with a yeast cell, all touching each other, which forced an error in our decision algorithm as marked in (d). The details of the 2D
correlation maps that are used for the decision making are further illustrated in Figure 5 for each cell/micro-object type. [Color figure can be seen in the online version of this article,
available at www.interscience.wiley.com.]

that is, the center of the cell shadow is darker, whereas the
center of the bead shadow is brighter with respect to the
background. This contrast reversal provides a powerful
means for automated separation of cells from other micro-
particles found within the same solution. Second, note also
that depending on the size of the particle, the strength of this
contrast reversal also varies (see e.g., top row of Fig. 2),
illustrating the uniqueness of the LUCAS signature of
different micro-objects. Third, due to its disk shape, the
shadow signature of the RBC shows a uniquely different
diffraction pattern (i.e., 2D texture) than other cells or
micro-beads. These results illustrate the fact that different
cell types (or micro-objects) have uniquely different
signatures (i.e., diffraction patterns, shadow shapes, and
contrast properties) in their LUCAS images, and this
important feature of LUCAS can be utilized by an
automated computer algorithm (i.e., the decision algo-
rithm) to characterize a heterogeneous mixture of cells/
micro-objects, even if the resolution level of the acquired
image is quite coarse (see e.g., Figs. 4 and 5).
As described in the previous section, the ultra-wide field
of view of the LUCAS platform stems from the fact that there
is no real magnification in LUCAS; and that the diffraction
pattern of each micro-particle is under-sampled by a large
area sensor array creating a pixilated signature for each
micro-particle type. Along these lines, Figures 1–5 illustrate
the proof-of-principles of LUCAS and its quantitative
comparison to conventional optical microscopy. On the
other hand, the exciting potential of LUCAS is not limited
only to the above results. Another exciting feature of the
LUCAS platform is that it permits monitoring, character-
ization and three-dimensional localization of different
microscopic objects within a much longer depth-of-field
(DOF) than a regular wide-field optical microscope would
normally permit. Therefore, in LUCAS various layers of
micro-objects (such as cells located within a multi-layered

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Figure 5. The 2D correlation maps for RBCs, yeast cells and 3 mm beads are
illustrated for the LUCAS experiment shown in Figure 4. Notice in (b and c) that the
characterization is accurate even if there is a partial overlap in the diffraction
signatures of some of the micro-objects/cells. [Color figure can be seen in the online
version of this article, available at www.interscience.wiley.com.]
862 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
microfluidic device; within a stack of microscope slides or
within a solution reservoir) can be monitored all in parallel
without the need for refocusing. In principle, LUCAS can
permit a DOF of
0.4 cm within which the diffraction
signature of each micro-object located at any given plane
exhibits a uniquely different pattern.
To experimentally verify this claim, we initially prepared
polystyrene micro-beads of different sizes (5, 10, and 20 mm
in diameter, Duke Scientific, Fremont, CA) diluted in 1
PBS. Then, using the LUCAS system, we imaged a multi-
layer structure as shown in Figure 6a. This multi-layered
object is composed of three different planes of micro-beads
separated by glass slides of a few hundreds of micrometer
thicknesses. For this multi-layered object, the total DOF was

0.85 mm, which is beyond the reach of a conventional
optical microscope, that is, mechanical adjustment of the
focus of the microscope would normally be needed to
monitor all the micro-beads located at different planes. This
limitation of conventional optical microscopy (under a 10
objective-lens) is verified in Figure 6d–f, where in each frame
only one type of micro-bead (located at a single plane) could
be imaged. As a result of its small DOF, three consecutive
microscope images were acquired (Fig. 6d–f) using a 10
objective-lens to image all the micro-beads located at
different planes within a DOF of
0.85 mm. On the
contrary, with LUCAS, a single lensless image captured the
uniquely different diffraction signatures of each type of
micro-particle, enabling monitoring and sorting of all the
micro-objects within the entire DOF.
Figure 6b shows the LUCAS image of this multi-layered
object, where the zoomed region (the white frame in
Figure 6b) is also highlighted in Figure 6c, illustrating the
location of different sized micro-beads with different
colored circles. The characteristic diffraction signature of
each micro-bead type that is located at a different plane as
depicted in Figure 6a is also shown in Figure 6g–i. The
comparison of the LUCAS image (Fig. 6c) with three
consecutive microscope images (Fig. 6d–f) corresponding to
the same region of interest at different image planes verifies
the stimulating potential of the LUCAS platform such that
various types of micro-particles within a large volume
(corresponding to a long DOF and a wide FOV) can be
monitored all in parallel.
To further investigate the performance of the LUCAS
platform for multi-layered objects, we conducted a series of
imaging experiments to build a small ‘‘shadow signature
library’’ corresponding to diffraction patterns of various
micro-objects at different plane heights. Figure 7a shows this
shadow signature library for various sized micro-beads
(D ¼ 5, 10, 20, and 30 mm) located at different plane heights
(S ¼ 0.30, 0.65, 1.00, and 4.10 mm) where D and S refer to
the diameter of the micro-beads, and the vertical distance
between micro-beads and the sensor array, respectively.
Therefore, S ¼ 0 defines the plane of the sensor array. And
each entry in Figure 7a has two images corresponding to two
different objects of the same diameter value, aimed to
demonstrate the uniformity of the acquired shadow
Figure 6. a: A multi-layered object that is composed of 3 planes of micro-beads (5, 10, and 20 mm diameter) over a depth-of-field of
0.85 mm. b: LUCAS image acquired for the
object shown in (a). c: Zoomed version of the LUCAS image taken from the white frame within (b). Each micro-object exhibits a uniquely different diffraction pattern, and the solid
red, dotted yellow and dashed blue circles represent 20, 10, and 5 mm diameter beads, respectively. A single LUCAS image, as shown in (b and c), shows all the particles with
distinct patterns within a dept-of-field of
1 mm and over a FOV of 10 cm2. On the other hand, a conventional microscope requires three consecutive images to monitor all these
different planes of micro-objects. d–f: Demonstrate three microscope images (taken with a 10 microscope objective) that illustrate the individual planes of the multi-layered object
shown in (a). Note that in each one of these microscope images, the other micro-particles are missing due to the limited depth-of-field of the microscope. g–l: The zoomed versions
of the LUCAS images and the microscope images (under 40) corresponding to 5, 10, and 20 mm diameter beads. [Color figure can be seen in the online version of this article,
available at www.interscience.wiley.com.]

signatures for a given micro-particle type. Each column in
Figure 7a illustrates that as the height of the micro-particle
increases, due to diffraction, its shadow signature spreads.
This implies that individual particles that share the same
diameter, shape and refractive index can all be localized
within a DOF of
4 mm by simply observing the shadow
size at the sensor plane. In other words, the height location
of each particle type within the sample volume is uniquely
encoded in its shadow width and texture. On the other hand,
each row in Figure 7a illustrates that the shadow signature of
different types of micro-particles at the same plane height all
exhibits similar shadow widths. However, this does not pose
a limitation for identification and localization of different
types of micro particles located at the same plane. Since the
contrast properties and the signal strength of these shadow
signatures corresponding to different particle types are all
uniquely different than each other, unambiguous identifica-
tion of each particle type located at the same plane is feasible.
Figure 7b–e shows the cross-sectional amplitude variations
in LUCAS shadow signatures corresponding to 5, 10, 20, and
30 mm sized beads at different plane heights, respectively.
Therefore, the results of Figure 7 indicate that based on the
unique size, shape, contrast and the signal strength of the
recorded shadow signatures, three-dimensional localization
and identification of different micro-particles within a
heterogeneous solution is possible with LUCAS.
To further explore the feasibility of multi-layer LUCAS
characterization, in Figure 8 we illustrate the results of a
LUCAS experiment for a two-layered structure that extends
over a DOF of
3.4 mm. Each layer in this experiment
contains a heterogeneous mixture of RBCs, yeast cells
(S. Pombe) and 10 mm beads located at planes S ¼ 1.2 mm
and S ¼ 3.4 mm (see Fig. 8). Figure 8b shows the raw LUCAS
image corresponding to this two-layered structure, and
Figure 8c shows a zoomed region of interest, where the
automated characterization results of the LUCAS decision
algorithm are illustrated. To point to the details of this
decision making process, similar to Figure 5, we also show in
Figure 9 the 2D correlation map of each micro-object type
located at both of the object planes. Figure 9 clearly
illustrates that using the LUCAS library image of the target
cell/micro-object type, an automated correlation analysis of
the LUCAS images (as discussed in the Materials and
Methods Section) yields unique correlation peaks for each
target object type. As illustrated in Figure 9, based on these
correlation maps corresponding to each target micro-object

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Figure 7. a: LUCAS signature library of various micro-beads (D ¼ 5, 10, 20, and 30 mm) located at different planes (S ¼ 0.30, 0.65, 1.00, and 4.10 mm). Each specific entry shows
two images to illustrate the uniformity of the acquired shadow signatures. b–e show the cross-sectional amplitude variations in 5 mm (b), 10 mm (c), 20 mm (d) and 30 mm
(e) diameter beads, respectively. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

type, unambiguous recognition of the diffraction signatures
of cells/particles located at different DOF planes is feasible.
Notice also that in Figure 9 the noise floor for the longer
DOF (S ¼ 3.4 mm) is higher for all the micro-objects when
compared to the shorter DOF plane (S ¼ 1.2mm). The main
reason for this behavior is the reduced contrast and SNR
in each LUCAS shadow due to increased effect of diffraction.
However, the correlation peaks of the target objects with the
LUCAS library image is still quite strong, yielding to the
correct decision regardless of the increased noise floor at
longer DOF values.
We should emphasize here that for a multi-layered sample
of interest that is imaged using LUCAS, some of diffraction
patterns of the particles/cells on different layers might
statistically overlap with each other causing interference in
their corresponding LUCAS pattern on the sensor array.
This interference could result in a characterization error as
the correlation of the interfering pattern with the LUCAS
library images would now be degraded. This overlap
problem could be especially severe for a high density of
cells. However, as briefly discussed earlier, for a sensor array
that has >10,000,000 pixels, a statistical error rate of <10%
can be achieved for a uniformly distributed heterogeneous
cell solution of 50,000 cells (total for all the layers of
interest).
Discussion
In the large depth-of-field LUCAS imaging results illustrated
above, different object planes were separated from each
other by 100 mm, forming the multi-layered object such as
in Figure 8. If this physical separation between different
object planes were to be reduced to <50 mm, the under-
sampled diffraction patterns corresponding to the same
micro-particle type located at adjacent planes would be quite
similar to each other, not only in 2D texture but also in
signature strength and contrast. This can potentially cause
errors in identifying the depth of the plane for a given micro-
object type within the sample volume. One possible solution
to this issue could be the use of a smaller pixel size sensor
array which can improve the spatial sampling of minute
differences in the diffraction patterns of adjacent planes. On
the other hand, this approach will ultimately reduce the field

864 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
Figure 8. The results of an automated LUCAS characterization of a heterogeneous mixture of RBCs, yeast cells and 10 mm beads imaged over a DOF of
3.4 mm are
illustrated. a: The same heterogeneous mixture is loaded on two different planes (S ¼ 1.2 mm and S ¼ 3.4 mm) and imaged using LUCAS. b: LUCAS raw image of this two layered
mixture is shown. c: The results of the LUCAS decision algorithm are illustrated. The unique diffraction signature of each RBC, yeast cell and microbead located at two different
layers (separated by >2 mm) is identified automatically. The details of the 2D correlation maps that are used for the decision making are illustrated in Figure 9 for each cell/micro-
object type. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

of view of the LUCAS platform, as it will demand more
pixels per diffraction signature of each micro-object/cell.
A more complete solution for improving the localization
accuracy in vertical (i.e., DOF) direction would be to utilize
angled illumination; that is, by changing the angle of
illumination of the LUCAS platform, the 2D pattern of
different micro-objects at different plane heights will shift
different amounts depending on the height of the object
plane from the sensor array. For instance the 2D signatures
of the micro-particles close to the sensor plane will shift
within the LUCAS image much less than other micro-
particles of the same kind that are located at higher depth of
field values. This multi-angle illumination LUCAS platform
may therefore provide an additional degree of freedom to
existing parameters (such as texture, width and signature
strength/contrast) to further improve the depth localization
accuracy of the LUCAS algorithm.
The diffraction pattern of each cell type strictly depends
on the illumination conditions such as the wavelength,
spatial coherence and object plane height. However, under
fixed illumination conditions, the 2D texture of the
diffraction signature of a given cell type physically depends
on three quantities: (i) the 3D morphology of the cell; (ii)
the refractive indices of the cell and its surrounding
medium; and (iii) the size of the cell. This directly implies
that during apoptosis, since cells exhibit unique morphology
changes (such as significant shrinkage), the LUCAS platform
can potentially discriminate vital cells from apoptotic cells
based on the variation of the classical diffraction pattern of
the cell. However, we should emphasize that not all cell types
would exhibit a detectable shadow signature difference
for automated characterization. The same is also true for
conventional lens based microscopy, that is, some cells look
quite the same even under high optical magnification. In
earlier sections we gave examples where the LUCAS shadow
signature of different cell lines and other particles were
uniquely different from each other and could be auto-
matically characterized using the LUCAS decision algo-
rithm. However, there are still various other cell types
(e.g., CD4 vs. CD8 cells among others) which share quite
similar morphology and refractive index values, as a result of
which their LUCAS signatures would yield comparable
shadow patterns making their automated characterization
rather difficult. To address this specificity issue of the
LUCAS platform for such similar looking cell types one
can follow the following methodologies: (1) dielectric
microbead labeling, (2) fluorescent labeling; and (3) surface
chemistry.

Su et al.: High-Throughput Lensfree Imaging On Chip 865

Biotechnology and Bioengineering
Figure 9. The 2D correlation maps are illustrated for the LUCAS experiment shown in Figure 8. The left panel shows S ¼ 1.2 mm plane whereas the right plane shows the
S ¼ 3.4 mm plane. For each plot, the LUCAS library image of the target micro-object is also shown at the bottom. Notice that the noise floor for the longer DOF (S ¼ 3.4 mm) is higher
for all the micro-objects; however, the correlation peaks of the target objects with the LUCAS library image is quite strong, yielding to the correct decision regardless of the
increased noise floor. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

Dielectric Microbead Labeling in LUCAS
Figure 2 clearly indicates that a polystyrene microbead
(regardless of its diameter) exhibits an opposite contrast
when compared to other cell lines. This contrast reversal
provides the basis for dielectric microbead based specific
labeling of a target cell type for significantly varying its
LUCAS signature. Therefore, a target cell type can be
specifically imaged within a heterogeneous cell solution by
using polystyrene microbeads that are coated with an
antibody corresponding to the CD marker of the target cell
type. Because target cells that are labeled with the microbead
will now exhibit a uniquely different shadow texture when
compared to other cell types within the same solution, the
specificity of the LUCAS platform will be significantly
improved.
Fluorescent Labeling in LUCAS
Another approach that one can utilize to bring molecular
specificity to LUCAS is to use fluorescent labeling. For this
purpose, we can use fluorescent dyes that are conjugated
with antibodies of a specific glycoprotein on the cell
membrane. This way, within a solution containing many
different types of cells, we can specifically label a target cell

866 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
type. To selectively read out the fluorescence signal coming
from dye molecules, we can use thin film filters deposited
onto the active region of the sensor array or at the bottom of
the used microfluidic channel, which selectively transmit
only the fluorescence signal. Thus, using such thin-film
filters, we can record LUCAS images of only the labeled cells.
After the acquisition of these fluorescent LUCAS images, we
can digitally compute the specific count of the labeled cells by
utilizing the LUCAS decision algorithm, which will also be
trained for similar lensfree fluorescence images using
homogenous solutions of the same labeled cell type. This
approach is quite different than the previously reported
fluorescence based cell counting technologies since it offers
a lensless on-chip platform, and is therefore much simpler
and easier to miniaturize. Further, ultra-wide FOV of
LUCAS allows analyzing much larger volume (
4 mL) in a
single test, significantly increasing the throughput of
characterization.
Use of Label-Free Surface Chemistry in LUCAS
A third alternative approach that can be used to bring
molecular specificity to LUCAS is the use of surface
chemistry based microfluidic devices. Such label-free
microfluidic technologies have been shown to have cell
capture efficiency and specificity of >90% and >85%,
respectively (Cheng et al., 2005, 2007). The specificity of this
approach is due to antibody coating of the inner surface of
the microfluidic channels. To avoid nonspecific capture of
other cell types that also express the target CD proteins, a
controlled flow rate through the microfluidic channels can
also be used. By controlling the flow rate, one can create an
optimum shear stress within the channels, which can break
the bonds of non-specifically captured cells before the target
cells’ bonds are broken. This microfluidic technology can
be integrated with LUCAS platform in a compact, self-
contained unit to measure the count of the captured cells
within the microfluidic chips without the use of any labeling
agents.
For the same cell type that are obtained from different
sources (such as RBCs obtained from two different healthy
donors), the overall LUCAS signatures showed very similar
properties as the above mentioned physical parameters
would on average be the same regardless of the source of
the cells of the same kind. As far as the passage number of the
cells is concerned, since for high passage numbers the
morphology of the cell can potentially start to deform, its
statistical LUCAS signature may deviate from the low
passage number of the same cell kind. However, such effects
should only be visible for ‘‘unhealthy cells’’ corresponding to
a large passage number, and can potentially be minimized by
independently monitoring the state of the cell population
through other means such as the protein expression level.
The experimental results presented in this manuscript
were acquired using floating/suspended cells, that is, the
imaged cells were not attached to a substrate. Note also that
adherent growing cell types such as fibroblasts that are
imaged using LUCAS were all detached from the substrate
during imaging. However, for imaging/monitoring of
surface adhered cells, the effect of the surface properties
of the substrate on the shadow image should also be
investigated. For this end, a new training set for each
adherent cell type for a given substrate needs to be acquired
to create a statistical image library for each cell-substrate
combination. This statistical LUCAS image library can then
be used to fine tune the decision algorithm for identification
of each adherent cell type for a given substrate. We should
note that a more detailed analysis of a larger scale LUCAS
signature library corresponding to various other cell types
(both adherent and non-adherent), together with a
discussion on the effect of the optical properties of the
LUCAS substrate and illumination wavelength on the image
signal-to-noise ratio and the LUCAS shadow signature is
currently under investigation and will be provided in a
further study.
Conclusions
In summary, we described a lensfree high-throughput on-
chip imaging platform that can potentially characterize

50,000 cells within a heterogeneous cell mixture over
depth-of-field of
4 mm and a field-of-view of
10 cm2.
We illustrated that the unique diffraction signatures of
different cell types and other micro-particles may enable
unambiguous automated characterization of a heteroge-
neous solution of
4 mL volume. For high-throughput cell
counting and characterization applications, where the main
aim is to rapidly count and identify cells over a large volume
or to dynamically monitor the location of thousands of cells,
LUCAS may provide numerous advantages over conven-
tional microscopes, such as a significantly increased sample
volume, reduced complexity, and ease of miniaturization.
Furthermore, LUCAS can also be integrated with micro-
fluidic channels providing massively parallel on-chip
monitoring, counting and characterization of cells without
using expensive and bulky optical components. This
technology may have a significant impact especially for
the developing world, where the resources are scarce, by
enabling a point-of-care cell counting device as small as a
regular cell-phone that uses for example, surface-chemistry
based micro-fluidic devices or fluorescent markers to test
the blood samples of patients with minimum amount of
reagent use.
References
Burger D, Gershman R. 1988. Acoustooptic laser scanning cytometer.
Cytometry 9:101–110.
Cheng X, Irimia D, Dixon M, Ziperstein JC, Demirce U, Zamir L, Tompkins
RG, Toner M, Rodriguez WR. 2005. A microchip approach for practical
Su et al.: High-Throughput Lensfree Imaging On Chip 867
Biotechnology and Bioengineering
 label-free CD4þ T-Cell counting of HIV-Infected subjects in resource-
poor settings. J. Acquir Immune Defic Syndr 45:257–261.
Cheng X, Irimia D, Dixon M, Ziperstein JC, Demirce U, Zamir L, Tompkins
RG, Rodriguez W, Toner M. 2007. A microfluidic device for practical
label-free CD4þ T cell counting of HIV-infected subjects. Lab Chip 7:
170–178.
Chin CD, Linder V, Sia SK. 2006. Lab-on-a-chip devices for global health:
Past studies and future opportunities. Lab Chip 7:41–57.
Darzynkiewicz Z, Elzbieta B, Li X, Gorczyca W, Myron Melamed MR. 1999.
Laser-scanning cytometry: A new instrumentation with many applica-
tions. Exp Cell Res 249:1–12.
Hulett HR, Bonner WA, Sweet RG, Herzenberg LA. 1973. Development and
application of a rapid cell sorter. Clin Chem 19:813–816.
Joo S, Chung TD, Kim HC. 2007. A rapid field-free electroosmotic micro-
pump incorporating charged microchannel surfaces. Sens Actuators B
Chem 132:1161–1168.
Kamei T, Paegel BM, Scherer JR, Skelley AM, Street RA, Mathies RA. 2003.
Integrated hydrogenated amorphous Si photodiode detector for micro-
fluidic bioanalytical devices. Anal Chem 75:5300–5305.
Kamentsky LA, Burger DE, Gershman RJ, Kamentsky LD, Luther E. 1997.
Slide-based laser scanning cytometry. Acta Cytol 41:123–143.
Lancaster C, Kokoris A, Nabavi M, Clemmens J, Maloney P, Capadanno J,
Gerdes J, Battrell CF. 2005. Rare cancer cell analyzer for whole blood
applications: Microcytometer cell counting and sorting subcircuits.
Methods 37:120–127.
Lee JS. 1981. Speckle analysis and smoothing of synthetic aperture radar
images. Comput Graph Image Process 17:24–32.
868 Biotechnology and Bioengineering, Vol. 102, No. 3, February 15, 2009
Lee JW. 2003. Global health improvement and WHO: Shaping the future.
Lancet 362:2083–2088.
Lien V, Zhao K, Berdichevsky Y, Lo YH. 2005. High-sensitivity cytometric
detection using fluidic-photonic integrated circuits with array wave-
guides. IEEE J Sel Top Quantum Electron 11:827–834.
Macnab RM, Koshland DE. 1972. The gradient-sensing mechanism in
bacterial chemotaxis. Proc Natl Acad Sci 69:2509–2512.
Niehren S, Kinzelbach W, Seeger S, Wolfrum J. 1995. An all-solid-state flow
cytometer for counting fluorescent microspheres. Anal Chem 67:2666–
2671.
Novak L, Neuzil P, Pipper J, Zhang Y, Lee SH. 2007. An integrated
fluorescence detection system for lab-on-a-chip applications. Lab Chip
6:27–29.
Ozcan A, Bilenca A, Bouma BE, Tearney GJ. 2006. Mirror tunnel micro-
scope. Appl Phys Lett 89:131124.
Ozcan A, Demirci U. 2007. Ultra wide-field lens-free monitoring of cells on-
chip. Lab Chip 8:98–106.
Steinberg DM, Ali SZ. 2001. Application of virtual microscopy in clinical
cytopathology. Diagn Cytopathol 25:389–396.
Weinstein R, Descour M, Liang C, Barker G, Scott K, Richter L, Krupinski E,
Bhattacharyya A, Davis J, Graham A. 2004. An array microscope for
ultra-rapid virtual slide processing and telepathology. Design, fabrica-
tion, and validation study. Hum Pathol 35:1303–1314.
Wolff A, Nielsen P, Larsen UD, Friis P, Goranovic G, Poulsen CR, Kutter JP,
Telleman P. 2003. Integrating advanced functionality in a microfab-
ricated high-throughput fluorescent-activated cell sorter. Lab Chip
3:22–27.